SAGE
Serial analysis of gene expression (SAGE) is a genetic technique used to directly quantify measurements of gene expression by isolating unique sequence tags (9-10 bp) from mRNAs and incorporating them into long DNA molecules for subsequent lump-sum sequencing (Yamamoto, Wakatsuki, Hada, & Ryo, 2001). An array of versions exist, including LongSAGE (Saha et al., 2002), RL-SAGE (Gowda, Jantasuriyarat, Dean, & Wang, 2004), and more recently, SuperSAGE (Matsumura et al., 2005). History Back in 1979 at Harvard and Caltech, research was pursuing the basic idea of in vitro synthesis of DNA copies of messenger RNAs. The research resulted in amplification of an entire library of such bacterial plasmids (Sim et al., 1979) and marks the basis for the development of SAGE. Shortly thereafter, from 1982 to 1983, a man named Greg Sutcliffe and his coworkers investigated the possibility of random (or semi-random) selection of clones from a cDNA library for sequencing (Sutcliffe, Milner, Bloom, & Lerner, 1982), followed by Putney who sequenced nearly 200 clones from a derived rabbit muscle cDNA library (Putney, Herlihy, & Schimmel, 1983). After about a decade, further progression towards SAGE was initiated by Adams who, with his coworkers, defined Expressed Sequence Tags (ESTs) (Adams et al., 1991) which now consist of approximately 72.6 million that are available in public databases such as GenBank. During the year of 1995, in an attempt to lower costs of mRNA surveys, Velculescu and coworkers investigated the length of these ESTs and derived shortened versions from about 100-800bp down to about 10-22bp (Velculescu, Zhang, Vogelstein, & Kinzler, 1995). These shortened tags represent the characteristic tagging method of SAGE. Techniques #mRNA preparation #cDNA synthesis #Cleavage of biotinylated cDNA with anchoring enzyme #Binding bitinylated cDNA to magnetic beads #Ligating linkers to bound cDNA #Release of cDNA tags using Tagging Enzyme #Blunt ending released cDNA tags #Ligating tags to form ditags #PCR amplification of ditags #Isolation of ditags #Purification of ditags #Ligation of ditags to form concatemers #Cloning concatemers and sequencing (SageNet Protocol, 2000) Applications SAGE is a common method of expression profiling (along with microarray analysis) (Horan, 2009; Mocellin & Rossi, 2007) due to its advantages such as identification power of fine variations in expression levels along with its ability to novel transcripts without any knowledge of the gene sequence (Liu et al., 2011). Any biological phenomena that result from changes in cellular transcription can be explored with SAGE application, providing a broad application basis. SAGE is also considered a highly competent technology that allows derivation of not only global gene expression but also gene expression of particular cell types and tissues. This competence assists in the identification of a set of specific genes to the cellular conditions by profile comparison (Yamamoto et al., 2001). The accessibility of SAGE data is also a huge benefit resulting from a multitude of biological applications. This data can be easily exchanged between laboratories and an immense database of SAGE data currently exists on the internet (van Ruissen & Baas, 2007). For more information regarding experimentally-specific applications of SAGE visit SAGENet.org. References Adams, M. D., Kelley, J. M., Gocayne, J. D., Dubnick, M., Polymeropoulos, M. H., Xiao, H., … Moreno, R. F. (1991). Complementary DNA sequencing: expressed sequence tags and human genome project. Science (New York, N.Y.), 252(5013), 1651–1656. Gowda, M., Jantasuriyarat, C., Dean, R. A., & Wang, G.-L. (2004). Robust-LongSAGE (RL-SAGE): a substantially improved LongSAGE method for gene discovery and transcriptome analysis. Plant physiology, 134(3), 890–897. doi:10.1104/pp.103.034496 Horan, M. P. (2009). Application of serial analysis of gene expression to the study of human genetic disease. Human genetics, 126(5), 605–614. doi:10.1007/s00439-009-0719-5 Liu, Y., Munro, D., Layfield, D., Dellinger, A., Walter, J., Peterson, K., … Hauser, M. (2011). Serial analysis of gene expression (SAGE) in normal human trabecular meshwork. Molecular Vision, 17, 885–893. Matsumura, H., Ito, A., Saitoh, H., Winter, P., Kahl, G., Reuter, M., … Terauchi, R. (2005). SuperSAGE. Cellular microbiology, 7''(1), 11–18. doi:10.1111/j.1462-5822.2004.00478.x Mocellin, S., & Rossi, C. R. (2007). Principles of gene microarray data analysis. ''Advances in experimental medicine and biology, 593, 19–30. doi:10.1007/978-0-387-39978-2_3 Putney, S. D., Herlihy, W. C., & Schimmel, P. (1983). A new troponin T and cDNA clones for 13 different muscle proteins, found by shotgun sequencing. Nature, 302(5910), 718–721. Saha, S., Sparks, A. B., Rago, C., Akmaev, V., Wang, C. J., Vogelstein, B., … Velculescu, V. E. (2002). Using the transcriptome to annotate the genome. Nature biotechnology, 20(5), 508–512. doi:10.1038/nbt0502-508 Sim, G. K., Kafatos, F. C., Jones, C. W., Koehler, M. D., Efstratiadis, A., & Maniatis, T. (1979). Use of a cDNA library for studies on evolution and developmental expression of the chorion multigene families. Cell, 18(4), 1303–1316. Sutcliffe, J., Milner, R., Bloom, F., & Lerner, R. (1982). Common 82-nucleotide sequence unique to brain RNA. Proceedings of the National Academy of Sciences of the United States of America, 79(16), 4942–4946. Van Ruissen, F., & Baas, F. (2007). Serial Analysis of Gene Expression. Methods in Molecularbiology, 383, 41–66. Velculescu, V. E., Zhang, L., Vogelstein, B., & Kinzler, K. W. (1995). Serial analysis of gene expression. Science (New York, N.Y.), 270(5235), 484–487. Yamamoto, M., Wakatsuki, T., Hada, A., & Ryo, A. (2001). Use of serial analysis of gene expression (SAGE) technology. Journal of immunological methods, 250(1-2), 45–66.